Baculovirus Transfer Vectors

Possee, Robert D.; King, Linda A.

doi:10.1007/978-1-4939-3043-2_3

Cited by 14 publications

(4 citation statements)

References 35 publications

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“…The full-length coding sequence was amplified because of the therapeutic potential of the completed protein purified from Nicotiana tobacum callus cells that exhibited neuroprotective effects. The amplified product was ligated into the pOET 1N_6X His transfer vector 23 . The purified plasmid was transformed into DH5α cells.…”

Section: Resultsmentioning

confidence: 99%

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Molecular Cloning and Expression of Osmotin in a Baculovirus-Insect System: Purified Osmotin Mitigates Amyloid-beta Deposition in Neuronal Cells

Abid

Yoon

Kim

2017

Sci Rep

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Osmotin is a pathogenesis-related plant protein, have gained focus of research because of its homology with mammalian adiponectin. The therapeutic properties of osmotin have been explored in recent years as it exhibits neuroprotective effects against amyloid beta-, glutamate- and ethanol-induced synaptic dysfunction and neurodegeneration. In the present study, the full-length gene of the tobacco plant osmotin was cloned and expressed in the Sf9 insect cell line using the baculovirus expression system. In vitro analysis of purified Osmotin protein showed excellent cell viability, p-AMPK activation and a reduction in amyloid-beta deposition. Immunofluorescent analysis showed significant reduction in amyloid beta deposition in APP over expressing neuronal cells. Osmotin inhibited amyloid beta deposition by influencing expression of APP processing genes including APP, ADAM 10 and BACE 1. Purified Osmotin showed reduction in amyloid beta deposition in different in vitro models as well. Osmotin showed similar mechanism when compared with mammalian adiponectin in different in vitro models. The present method will be an excellent approach for the efficient and cost-effective production of the functional protein to be utilized for therapeutic purposes. Reduction in amyloid beta deposition by activation of p-AMPK influencing APP processing genes makes osmotin a potent therapeutic candidate for neurodegenerative diseases.

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Section: Resultsmentioning

confidence: 99%

“…Sf9 cells are easy to culture both in an adherent and suspension culturing condition. The 6x histidine tag was used in the extraction and purification of ultra-pure osmotin with the Ni-NTA purification system 23 .…”

Section: Methodsmentioning

confidence: 99%

Molecular Cloning and Expression of Osmotin in a Baculovirus-Insect System: Purified Osmotin Mitigates Amyloid-beta Deposition in Neuronal Cells

Abid

Yoon

Kim

2017

Sci Rep

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“…One of the main drawbacks of insect cell expression systems is the complexity of the setup compared to microbial systems in terms of facilities and biochemical tools needed to establish cell lines. However, over the last 30 years, insect cell expression systems have experienced a remarkable evolution, with new versatile and flexible tools and methods being developed ( Possee and King, 2016 ; Chambers et al, 2018 ).…”

Section: Systems For the Heterologous Expression Of Toxinsmentioning

confidence: 99%

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

Rivera-de-Torre

Rimbault

Jenkins

et al. 2022

Front. Bioeng. Biotechnol.

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Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

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“…Though not specifically covered in this unit, a key factor in protein expression success is the choice of promoter to drive gene expression (Possee & King, 2016). The polyhedrin (polh) promoter was the original and remains the most commonly used promoter (Smith et al, 1983), and drives a strong burst of expression with high protein levels expected from ß27 h.p.i.…”

Section: Critical Parameters Promoter/transfer Plasmid Designmentioning

confidence: 99%

Protein Production using the Baculovirus Expression System

et al. 2018

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Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production. We also cover the use of small-scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale-up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.

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Baculovirus Transfer Vectors

Cited by 14 publications

References 35 publications

Molecular Cloning and Expression of Osmotin in a Baculovirus-Insect System: Purified Osmotin Mitigates Amyloid-beta Deposition in Neuronal Cells

Molecular Cloning and Expression of Osmotin in a Baculovirus-Insect System: Purified Osmotin Mitigates Amyloid-beta Deposition in Neuronal Cells

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

Protein Production using the Baculovirus Expression System

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